Many commercially important polypeptides are produced in recombinant cells grown in culture. One method for producing such polypeptides involves the generation of an amplified cell line by transfection of host cells with two nucleic acid sequences, one of which encodes a polypeptide of interest, and another which encodes an amplifiable marker, such as dihydrofolate reductase (DHFR). In instances in which the amplifiable marker is DHFR, transfected cells are subjected to selective pressure by culturing the cells in increasing concentrations of the amplification agent methotrexate (MTX), as is well known in the art. The normally cytotoxic effects of MTX are substantially eliminated by expression of DHFR. Transfected cells are believed to survive because the DHFR gene has been integrated into the genome of the transfected cell. Increasing the DHFR copy number to a sufficiently high level through amplification enables the cell line to be resistant to higher levels of MTX. Similarly, as the nucleic acid sequence encoding DHFR is amplified, the nucleic acid sequence encoding the polypeptide of interest is also amplified, thereby increasing expression of that sequence.
However, this method of amplification and increased expression has drawbacks. In particular, the generation of most suitable recombinant cell lines utilizing this method is labor-intensive and can require several months to perform. For example, this amplification method has traditionally been performed in tissue culture flasks, shake flasks, or spinner flasks by subjecting a cell line with a low level of expression of the amplifiable marker to sequential step-wise increases in concentration of the amplification agent after maintaining the cells for several weeks. Thus, the time required to progress to the next stage can be several months. In addition, previous methods of amplification are typically labor-intensive because the cell lines must continuously be evaluated over several months to ensure that amplification is occurring.
Accordingly, it would be desirable to provide efficient methods for the amplification of cell lines that express a polypeptide of interest in a relatively short period of time.